Method of making an electrochemical sensor strip

ABSTRACT

A method of making an electrochemical sensor strip that includes: depositing a first electrode on a base; depositing a second electrode on the base; applying a first layer onto the first electrode; and applying a second layer onto the second electrode. The first layer includes an oxidoreductase and a mediator. The second layer includes a soluble redox species.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional applicationSer. No. 13/214,643, filed Aug. 22, 2011, entitled “EnzymaticElectrochemical Biosensor”, which is a continuation of U.S.Nonprovisional application Ser. No. 10/576,485, filed Apr. 21, 2006,entitled “Enzymatic Electrochemical Biosensor”, now U.S. Pat. No.8,007,656, issued on Aug. 30, 2011, which was the National Stage ofInternational Application No. PCT/US04/35286, filed Oct. 22, 2004,entitled “Enzymatic Electrochemical Biosensor”, which was published inEnglish and claims the benefit of U.S. Provisional Application No.60/513,817, filed Oct. 24, 2003, entitled “Enzymatic ElectrochemicalBiosensor”.

BACKGROUND

In monitoring medical conditions and the response of patients to effortsto treat medical conditions, it is desirable to use analytical methodsthat are fast, accurate, and convenient for the patient. Electrochemicalmethods have been useful for quantifying certain analytes in bodyfluids, particularly in blood samples. Typically, these analytes undergooxidation-reduction reactions when in contact with specific enzymes, andthe electric current generated by these reactions can be correlated withthe concentration of the analyte of interest. Miniaturized versions ofanalytical electrochemical cells have been developed that allow patientsto monitor levels of particular analytes on their own, without the needfor a healthcare provider or clinical technician. Typicalpatient-operated electrochemical sensors utilize a single measuring unitcontaining the necessary circuitry and output systems. In use, this unitis connected to a disposable analysis strip containing the electrodesand the necessary reagents to measure the electrochemical properties ofa sample that is applied to the strip. The most common of theseminiature electrochemical systems are the glucose sensors that providemeasurements of blood glucose levels. Ideally, a miniature sensor forglucose should provide accurate readings of blood glucose levels byanalyzing a single drop of blood, typically from 1-15 microliters (μL).

In a typical analytical electrochemical cell, regardless of the size ofthe system, the oxidation or reduction half-cell reaction involving theanalyte either produces or consumes electrons. This electron flow can bemeasured, provided the electrons can interact with a working electrodethat is in contact with the sample to be analyzed. The electricalcircuit is completed through a counter electrode that is also in contactwith the sample. A chemical reaction also occurs at the counterelectrode, and this reaction is of the opposite type (oxidation orreduction) relative to the type of reaction at the working electrode.See, for example, Fundamentals Of Analytical Chemistry, 4^(th) Edition,D. A. Skoog and D. M. West; Philadelphia: Saunders College Publishing(1982), pp 304-341.

In some conventional miniature electrochemical systems used fordiagnostics, a combination counter/reference electrode is employed. Thistype of combination electrode is possible when the reference electrodematerials are separated, by their insolubility, from the reactioncomponents of the analysis solution. Counter/reference electrodes aretypically a mixture of silver (Ag) and silver chloride (AgCl), whichexhibits stable electrochemical properties due to the insolubility ofits components in the aqueous environment of the analysis solution.Since the ratio of Ag to AgCl is not significantly changed during use,the electrochemical properties of the electrode are likewise notsignificantly changed.

Although the Ag/AgCl electrode functions well as a reference electrode,it can be less than ideal in its function as a counter electrode. TheAg/AgCl material has a high resistivity, which inhibits its capacity forcarrying electrical current. Thus, high voltages and/or current levelsmay be necessary to operate the sensor. This can be especiallyproblematic in miniaturized electrochemical analysis strips, since smalluncertainties and variabilities can dramatically reduce the sensitivityof the measurement. Samples containing high concentrations of theanalyte can yield erroneous results if the high current produced throughreaction of the analyte is impeded by the counter electrode.

Another feature of some conventional miniaturized electrochemical stripsis the presence of a single layer of reagents over both the working andcounter electrodes. The components of this reagent layer include theenzyme that facilitates the oxidation-reduction reaction of the analyte,as well as any mediators or other substances that help to transferelectrons between the oxidation-reduction reaction and the workingelectrode. The use of a single reagent layer can provide for simplemanufacturing of the strips, since only one deposition step coats thematerial onto the electrodes. A disadvantage of the single layerconstruction is that each electrode is in contact with the sameenvironment when the device is in use. Thus, the individual environmentof each electrode is not controlled to provide the optimum conditionsfor electrode function. This lack of optimization can also reduce thesensitivity of the system.

There is a need for miniaturized electrochemical systems with improvedsensitivity to the concentration of analytes in patient samples. It isdesirable for miniaturized electrochemical strips to containindependently optimized electrodes having high conductivities.

SUMMARY

In one aspect of the invention, there is an electrochemical sensorstrip, comprising a base; a first electrode on the base; a secondelectrode on the base; an oxidoreductase enzyme and a mediator on thefirst electrode; and a soluble redox species on the second electrode.

In another aspect of the invention, there is an electrochemical sensorstrip, comprising a base; a first electrode on the base; a secondelectrode on the base; an enzyme on the first electrode, where theenzyme is glucose oxidase, glucose dehydrogenase or a mixture thereof; amediator on the first electrode; and a soluble redox species on thesecond electrode. The soluble redox species is preferably anorganotransition metal complex, a transition metal coordination complexor mixtures thereof.

In yet another aspect of the invention, there is a method of making anelectrochemical sensor strip, comprising depositing a first electrode ona base; depositing a second electrode on the base; applying a firstlayer onto the first electrode, the first layer comprising anoxidoreductase and a mediator; and applying a second layer onto thesecond electrode, the second layer comprising a soluble redox species.

In yet another aspect of the invention, there is a method of quantifyingan analyte in a sample, comprising contacting the sample with anelectrochemical sensor strip; the electrochemical sensor stripcomprising a first electrode and a first layer on the first electrode,the first layer comprising an oxidoreductase enzyme and a mediator; theelectrochemical sensor strip also comprising a second electrode and asecond layer on the second electrode, the second layer comprising asoluble redox species; applying an electrical potential between thefirst and second electrodes; measuring a current passing through thefirst and second electrodes and the sample; and correlating the currentto a concentration of the analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top view diagram of a sensor base containing a workingelectrode and a counter electrode.

FIG. 2 is an end view diagram of the sensor base of FIG. 1.

FIG. 3 is a top view diagram of a sensor base and electrodes under adielectric layer.

FIG. 4 is a perspective view diagram of a completely assembled sensorstrip.

FIG. 5 is a photomicrograph of a working electrode and counter electrodehaving separate reagent layers.

FIG. 6 is a set of cyclic voltammograms for sensor strips havingdifferent layers on the counter electrode.

FIG. 7 is a graph of measured current as a function of glucoseconcentration for sensor strips having different counter electrodes.

FIG. 8 is a set of cyclic voltammograms for sensor strips havingdifferent working electrodes.

FIG. 9 is a set of cyclic voltammograms at various glucoseconcentrations for a sensor strip having ferricyanide on the counterelectrode, but with no ferrocyanide on the working electrode.

FIG. 10 is a graph of measured current as a function of glucoseconcentration for a sensor strip.

FIG. 11 is a set of cyclic voltammograms at various glucoseconcentrations for a sensor strip having 3-phenylimino-3H-phenothiazineon both the working and counter electrodes.

FIGS. 12-14 are top view diagrams of sensor bases containing a workingelectrode, a counter electrode, and a third electrode.

FIG. 15 is a cutaway view diagram of the sensor base of FIG. 13.

DETAILED DESCRIPTION

The present invention relates to an electrochemical biosensor fordetermining the presence or amount of a substance in a sample. Thebiosensor includes sensor strips containing a working electrode and acounter electrode, each of which is at least partially covered with aseparate reagent layer. The reagent layer on the working electrodeincludes an enzyme that interacts with an analyte through anoxidation-reduction reaction and also includes a mediator. The reagentlayer on the counter electrode includes a soluble redox species that canundergo the opposite type of oxidation-reduction reaction with respectto the analyte reaction. The soluble redox species is preferably presentin the counter electrode reagent layer in a molar amount greater thanthat of its counterpart species, where the redox species and itscounterpart species together are a redox pair. Sensors of the presentinvention may provide for improvements in accuracy, range of analysis,and shelf life.

The term “sample” is defined as a composition containing an unknownamount of the analyte of interest. Typically, a sample forelectrochemical analysis is in liquid form, and preferably the sample isan aqueous mixture. A sample may be a biological sample, such as blood,urine or saliva. A sample may be a derivative of a biological sample,such as an extract, a dilution, a filtrate, or a reconstitutedprecipitate.

The term “analyte” is defined as a substance in a sample, the presenceor amount of which is to be determined. An analyte interacts with theoxidoreductase enzyme present during the analysis, and can be asubstrate for the oxidoreductase, a coenzyme, or another substance thataffects the interaction between the oxidoreductase and its substrate.

The term “oxidoreductase” is defined as any enzyme that facilitates theoxidation or reduction of a substrate. The term oxidoreductase includes“oxidases,” which facilitate oxidation reactions in which molecularoxygen is the electron acceptor; “reductases,” which facilitatereduction reactions in which the analyte is reduced and molecular oxygenis not the analyte; and “dehydrogenases,” which facilitate oxidationreactions in which molecular oxygen is not the electron acceptor. See,for example, Oxford Dictionary of Biochemistry and Molecular Biology,Revised Edition, A. D. Smith, Ed., New York: Oxford University Press(1997) pp. 161, 476, 477, and 560.

The term “oxidation-reduction” reaction is defined as a chemicalreaction between two species involving the transfer of at least oneelectron from one species to the other species. This type of reaction isalso referred to as a “redox reaction.” The oxidation portion of thereaction involves the loss of at least one electron by one of thespecies, and the reduction portion involves the addition of at least oneelectron to the other species. The ionic charge of a species that isoxidized is made more positive by an amount equal to the number ofelectrons transferred. Likewise, the ionic charge of a species that isreduced is made less positive by an amount equal to the number ofelectrons transferred.

The term “oxidation number” is defined as the formal ionic charge of achemical species, such as an atom. A higher oxidation number, such as(III), is more positive, and a lower oxidation number, such as (II), isless positive. A neutral species has an ionic charge of zero. Oxidationof a species results in an increase in the oxidation number of thatspecies, and reduction of a species results in a decrease in theoxidation number of that species.

The term “redox pair” is defined as two species of a chemical substancehaving different oxidation numbers. Reduction of the species having thehigher oxidation number produces the species having the lower oxidationnumber. Alternatively, oxidation of the species having the loweroxidation number produces the species having the higher oxidationnumber.

The term “oxidizable species” is defined as the species of a redox pairhaving the lower oxidation number, and which is thus capable of beingoxidized into the species having the higher oxidation number. Likewise,the term “reducible species” is defined as the species of a redox pairhaving the higher oxidation number, and which is thus capable of beingreduced into the species having the lower oxidation number.

The term “soluble redox species” is defined as a substance that iscapable of undergoing oxidation or reduction and that is soluble inwater (pH 7, 25° C.) at a level of at least 1.0 grams per Liter. Solubleredox species include electroactive organic molecules, organotransitionmetal complexes, and transition metal coordination complexes. The term“soluble redox species” excludes elemental metals and lone metal ions,especially those that are insoluble or sparingly soluble in water.

The term “organotransition metal complex,” also referred to as “OTMcomplex,” is defined as a complex where a transition metal is bonded toat least one carbon atom through a sigma bond (formal charge of −1 onthe carbon atom sigma bonded to the transition metal) or a pi bond(formal charge of 0 on the carbon atoms pi bonded to the transitionmetal). For example, ferrocene is an OTM complex with twocyclopentadienyl (Cp) rings, each bonded through its five carbon atomsto an iron center by two pi bonds and one sigma bond. Another example ofan OTM complex is ferricyanide (III) and its reduced ferrocyanide (II)counterpart, where six cyano ligands (formal charge of −1 on each of the6 ligands) are sigma bonded to an iron center through the carbon atomsof the cyano groups.

The term “coordination complex” is defined as a complex havingwell-defined coordination geometry, such as octahedral or square planargeometry. Unlike OTM complexes, which are defined by their bonding,coordination complexes are defined by their geometry. Thus, coordinationcomplexes may be OTM complexes (such as the previously mentionedferricyanide), or complexes where non-metal atoms other than carbon,such as heteroatoms including nitrogen, sulfur, oxygen, and phosphorous,are datively bonded to the transition metal center. For example,ruthenium hexaamine is a coordination complex having a well-definedoctahedral geometry where six NH₃ ligands (formal charge of 0 on each ofthe 6 ligands) are datively bonded to the ruthenium center. A morecomplete discussion of organotransition metal complexes, coordinationcomplexes, and transition metal bonding may be found in Collman et al.,Principles and Applications of Organotransition Metal Chemistry (1987)and Miessler & Tarr, Inorganic Chemistry (1991).

The term “mediator” is defined as a substance that can be oxidized orreduced and that can transfer one or more electrons between a firstsubstance and a second substance. A mediator is a reagent in anelectrochemical analysis and is not the analyte of interest. In asimplistic system, the mediator undergoes a redox reaction with theoxidoreductase after the oxidoreductase has been reduced or oxidizedthrough its contact with an appropriate substrate. This oxidized orreduced mediator then undergoes the opposite reaction at the electrodeand is regenerated to its original oxidation number.

The term “electroactive organic molecule” is defined as an organicmolecule that does not contain a metal and that is capable of undergoingan oxidation or reduction reaction. Electroactive organic molecules canbehave as redox species and as mediators. Examples of electroactiveorganic molecules include coenzyme pyrroloquinoline quinone (PQQ),benzoquinones and naphthoquinones, N-oxides, nitroso compounds,hydroxylamines, oxines, flavins, phenazines, phenothiazines,indophenols, and indamines.

The term “electrode” is defined as an electrically conductive substancethat remains stationary during an electrochemical analysis. Examples ofelectrode materials include solid metals, metal pastes, conductivecarbon, conductive carbon pastes, and conductive polymers.

The term “non-ionizing material” is defined as a material that does notionize during the electrochemical analysis of an analyte. Examples ofnon-ionizing materials include carbon, gold, platinum and palladium.

The term “on” is defined as “above” and is relative to the orientationbeing described. For example, if a first element is deposited over atleast a portion of a second element, the first element is said to be“deposited on” the second. In another example, if a first element ispresent above at least a portion of a second element, the first elementis said to be “on” the second. The use of the term “on” does not excludethe presence of substances between the upper and lower elements beingdescribed. For example, a first element may have a coating over its topsurface, yet a second element over at least a portion of the firstelement and its top coating can be described as “on” the first element.Thus, the use of the term “on” may or may not mean that the two elementsbeing related are in physical contact with each other.

Electrochemical analytical sensors can be constructed in a variety ofways and using a variety of materials. See, for example, U.S. Pat. Nos.5,120,420 and 5,798,031, both of which are incorporated herein byreference. Referring to FIGS. 1-3 and 12-15, in general, electrodematerials are deposited onto a base material 10. The base material ispreferably an electrical insulator so as to isolate the electrochemicalsystem from its surroundings. The electrode materials can be configuredto connect to outside circuitry, for example through conductors 12 and14. These connections allow the electrochemical response of the sensorto be monitored and/or manipulated. Preferably, the electrode materialsand the entire sensor are configured to be compatible with existingelectroanalytical measurement devices. In addition, the electrodematerials are configured to provide contact with the sample to beanalyzed. Each electrode is coated with a layer of material containingappropriate analytical reagents. The working electrode 20 is coated witha first layer 26 containing an oxidoreductase enzyme and a mediator. Thecounter electrode 30 is coated with a second layer 36 containing asoluble redox species. The electrodes may be partially covered by adielectric layer 40, such as an insulating polymer, to isolate theportions of the electrodes that are in contact with the reagent layersfrom the portions that connect the electrodes to outside circuitry. Thisdielectric layer, if present, may be deposited before, during or afterthe coating of the electrodes with the appropriate reagent layer. Theentire assembly is then covered at least partially with a lid 50.Preferably the lid covers, but does not contact, the reagent layers, soas to providing a space for the sample to be deposited and analyzed.Optional third electrode 70 is coated with a third layer 76 containing asoluble redox species. The optional third electrode may be configured toconnect to outside circuitry through conductor 13.

Each electrode may contain any electrically conductive substance,including metals, conductive polymers, and conductive carbon. Examplesof conductive materials include a thin layer of a metal such as gold,silver, platinum, palladium, copper, or tungsten, as well as a thinlayer of conductive carbon powder. Preferably, electrodes that are incontact with the sample during the use of the sensor are made of inertmaterials, such that the electrode does not undergo a net oxidation or anet reduction during the analysis. More preferably, electrodes that arein contact with the sample during the use of the sensor are made ofnon-ionizing materials, such as carbon, gold, platinum, and palladium.In some instances, ionizing materials, such as silver, can form redoxspecies during the use of the sensor that can adversely influence themeasured current or potential of the system.

Metals may be deposited on a base material by deposition of a metalfoil, by chemical vapor deposition, or by deposition of a slurry of themetal on the base. Conductive carbon may be deposited, for example, bypyrolysis of a carbon-containing material or by deposition of a slurryof carbon powder. The slurry may contain more than one type ofconductive material. For example, the slurry may contain both palladiumand carbon powder. In the case of slurry deposition, the fluid mixturemay be applied as an ink to the base material, as described in U.S. Pat.No. 5,798,031.

The example illustrated in FIG. 2 shows a working electrode 20 and acounter electrode 30, each of which contain a main conductor 22 or 32and an optional surface conductor 24 or 34. The configuration and thenumber of components of a given electrode system can be widely varied tooptimize the electrical response of the electrodes to theelectrochemistry that takes place when a sample is being analyzed. Itmay be desirable for a main conductor (22 or 32) to be one end of aportion of a single conductive substance, while the connectingconductors (12 or 14) are at the other end of the portion. The optionalsurface conductor may then function to convert the electrochemicalsignal into solid-state electron flow to be communicated to a measuringunit via the conductive substance (i.e. flowing from 22 to 12). In oneexample, main conductors 22 and 32 are pieces of metal foil that arecontiguous with the connecting conductors 12 and 14, and the surfaceconductors 24 and 34 are layers of conductive carbon powder.

The example illustrated in FIG. 15 shows a counter electrode 30 and athird electrode 70, each of which contain a main conductor 32 or 72 andan optional surface conductor 34 or 74. The third electrode componentscan also be varied to optimize the overall performance of the sensor. Itmay be desirable for the main conductor 72 to be one end of a portion ofa single conductive substance, while the connecting conductor 13 is atthe other end of the portion. In one example, main conductor 72 is apiece of metal foil that is contiguous with connecting conductor 13.Optional surface conductor 74 may be, for example, a layer of conductivecarbon powder. The third layer 76, containing a soluble redox species,may have a composition that is different from the composition of thesecond layer 36, or the second and third layers may be identical. In oneexample, the third layer is a portion of the second layer that isconfigured to cover the conductors 32 (and optionally 34) and 72 (andoptionally 74).

If an electrode contains a surface electrode, it is preferred that thesurface electrode is a non-ionizing conductive material. If an electrodeis simply a layer of conductive material without a distinct conductinglayer on its surface, it is preferred that the conductive material isnon-ionizing. More preferably, the surface of the counter electrode,which may or may not be a layer distinct from the main conductor, is anon-ionizing material.

The reagent layers for the working and counter electrodes havecompositions that are different from each other. This distinctnessallows the reagent layers to be separately optimized to provide a sensorstrip having improved electrochemical analysis properties. The layer onthe working electrode may contain ingredients that facilitate thereaction of the analyte and the communication of the results of thisreaction to the electrode and the connected circuitry. The layer on thecounter electrode may contain ingredients that facilitate the free flowof electrons between the sample being analyzed and the electrode and itsconnected circuitry. To further optimize the system, a third electrodemay have a layer containing ingredients that facilitate the free flow ofelectrons between the sample being analyzed and the electrode and itsconnected circuitry. Each of the layers may independently contain inertingredients that are not directly involved in any oxidation-reductionreactions in the electrochemical cell. Examples of such inertingredients include binding agents such as bentone, polyethylene oxide,or carboxymethyl cellulose; thickening agents such as silica orpolyethylene oxide; and one or more buffers.

The layer on the working electrode preferably contains an oxidoreductaseenzyme. The oxidoreductase may be specific for a substrate that is theanalyte of interest. The oxidoreductase may be specific for a substratesuch that the reaction of the oxidoreductase and its substrate isaffected by the presence or amount of the analyte of interest. Examplesof oxidoreductases and their specific substrates are given in Table I.For example, an alcohol oxidase can be used in the reagent layer toprovide a sensor that is sensitive to the presence of alcohol in asample. Such a system could be useful in measuring blood alcoholconcentrations. In another example, glucose dehydrogenase or glucoseoxidase can be used in the reagent layer to provide a sensor that issensitive to the presence of glucose in a sample. This system could beuseful in measuring blood glucose concentrations, for example inpatients known or suspected to have diabetes. If the concentrations oftwo different substances are linked through a known relationship, thenthe measurement of one of the substances through its interaction withthe oxidoreductase can provide for the calculation of the concentrationof the other substance. For example, an oxidoreductase may provide asensor that is sensitive to a particular substrate, and the measuredconcentration of this substrate can then be used to calculate theconcentration of the analyte of interest.

TABLE I Oxidoreductase (reagent layer) Substrate/analyte Glucosedehydrogenase β-glucose Glucose oxidase β-glucose Cholesterol esterase;cholesterol oxidase Cholesterol Lipoprotein lipase; glycerol kinase;glycerol-3- Triglycerides phosphate oxidase Lactate oxidase; lactatedehydrogenase; Lactate diaphorase Pyruvate oxidase Pyruvate Alcoholoxidase Alcohol Bilirubin oxidase Bilirubin Uricase Uric acidGlutathione reductase NAD(P)H Carbon monoxide oxidoreductase Carbonmonoxide

The layer on the working electrode may contain one or more mediatorsubstances. The presence of a mediator can enhance the transmission ofelectrical signal from the enzyme-facilitated redox reaction to theelectrode material. Without wishing to be bound by any theory ofinterpretation, it is believed that mediators may act either as a redoxcofactor in the initial enzymatic reaction or as a redox collector toaccept electrons from or donate electrons to the enzyme or other speciesafter the reaction has occurred. In the situation of a redox cofactor,the mediator is believed to be the species that balances the redoxreaction of the substrate. Thus if the substrate is reduced, themediator is oxidized. In the situation of a redox collector, anotherspecies may have been oxidized or reduced initially to balance the redoxreaction of the substrate. This species may be the oxidoreductaseitself, or it may be another species such as a redox cofactor.

Mediators in enzymatic electrochemical cells are described, for examplein U.S. Pat. No. 5,653,863, which is incorporated herein by reference.In some cases, the mediator may function to regenerate theoxidoreductase. For example, if the enzyme oxidizes a substrate, theenzyme itself is reduced. Interaction of this enzyme with a mediator canresult in reduction of the mediator, together with oxidation of theenzyme to its original, unreacted state. Interaction of the mediatorwith the electrode at an appropriate electrical potential can result ina release of one or more electrons to the electrode together withoxidation of the mediator to its original, unreacted state.

Examples of mediators include OTM and coordination complexes, includingferrocene compounds such as 1,1′-dimethyl ferrocene; and includingcomplexes described in U.S. Pat. No. 5,653,863, such as ferrocyanide andferricyanide. Examples of mediators also include electroactive organicmolecules including coenzymes such as coenzyme pyrroloquinoline quinone(PQQ); the substituted benzoquinones and naphthoquinones disclosed inU.S. Pat. No. 4,746,607, which is incorporated herein by reference; theN-oxides, nitroso compounds, hydroxylamines and oxines specificallydisclosed in EP 0 354 441, which is incorporated herein by reference;the flavins, phenazines, phenothiazines, indophenols, substituted1,4-benzoquinones and indamines disclosed in EP 0 330 517, which isincorporated herein by reference; and the phenazinium and phenoxaziniumsalts disclosed in U.S. Pat. No. 3,791,988, which is incorporated hereinby reference. A review of electrochemical mediators of biological redoxsystems can be found in Analytica Clinica Acta. 140 (1982), pages 1-18.Examples of electroactive organic molecule mediators also include thosedescribed in U.S. Pat. No. 5,520,786, which is incorporated herein byreference, including 3-phenylimino-3H-phenothiazine (PIPT), and3-phenylimino-3H-phenoxazine (PIPO).

The reagent layer on the counter electrode contains a soluble redoxspecies. The soluble redox species undergoes the opposite reactionrelative to the reaction of the substrate of the oxidoreductase, and inso doing is converted into its counterpart species of the redox pair.For example, if the analyte is reduced, the soluble redox species isoxidized; and if the analyte is oxidized, the soluble redox species isreduced. The counterpart species of the redox pair may also be presentin the layer, but it is preferably present in a concentration lower thanthe concentration of the primary redox species. More preferably, theredox species in the reagent layer on the counter electrode isexclusively the soluble redox species that undergoes the oppositereaction relative to the reaction of the substrate of theoxidoreductase.

A soluble redox species may be an electroactive organic molecule, it maybe an organotransition metal complex, it may be a transition metalcoordination complex, or it may be mixtures of any of these. Forexample, organic molecules such as coenzyme pyrroloquinoline quinone(PQQ), substituted benzoquinones and naphthoquinones, N-oxides, nitrosocompounds, hydroxylamines, oxines, flavins, phenazines, phenothiazines,indophenols, indamines, phenazinium salts and phenoxazinium salts mayeach be a soluble redox species.

A soluble redox species may be an organotransition metal complex or atransition metal coordination complex. Many transition metals occurnaturally as compounds with hydrogen, oxygen, sulfur, or othertransition metals, and these transition metals are generally observed inone or more oxidation states. For example iron, chromium, and cobalt aretypically found in oxidation states of +2 (i.e. II) or +3 (i.e. III).Thus, iron (II) and iron (III) are two species of a redox pair. Manyelemental metals or metal ions, however, are only sparingly soluble inaqueous environments, limiting their utility as redox species inbalancing the oxidation-reduction reactions in an electrochemicalanalysis system. Metal ions that are bonded or coordinated to ligandscan be made more soluble by their association with those ligands.Typically, the metal in an organotransition metal complex or atransition metal coordination complex is the moiety in the complex thatis actually reduced or oxidized. For example, the iron center inferrocene [Fe(II)(C₅H₆)₂] and in the ferrocyanide ion [Fe(II)(CN)₆]⁴⁻ isin the +2 formal oxidation state, while the ferricyanide ion[Fe(III)(CN)₆]³⁻ contains iron in its +3 formal oxidation state.Ferrocyanide and ferricyanide together form a redox pair, and either onecan function as the soluble redox species in the reagent layer on thecounter electrode, depending on the type of oxidoreductase used on theworking electrode. An example of a redox pair containing transitionmetal coordination complexes is the combination of two species ofruthenium hexaamine, [Ru(III)(NH₃)₆]³⁺ and [Ru(II)(NH₃)₆]²⁺.

The species of the redox pair that is present in the reagent layer onthe counter electrode, referred to as the first species, is preferablypresent in a greater molar amount than its counterpart species (i.e. thesecond species) of the same redox pair. Preferably, the molar ratio ofthe first species to the second species is at least 1.2:1. Morepreferably, the molar ratio of the first species to the second speciesis at least 2:1. Still more preferably, the molar ratio of the firstspecies to the second species is at least 10:1. Still more preferably,the molar ratio of the first species to the second species is at least100:1. Still more preferably, the second species of the redox pair ispresent in an amount of 1 part per thousand (ppt) or less prior to theuse of the sensor strip in an analysis. Still more preferably, thesecond species of the redox pair is present in an amount of 1 part permillion (ppm) or less prior to the use of the sensor strip in ananalysis.

Preferably, the soluble redox species is solubilized in the sample andmixes with the analyte and other sample constituents. The soluble redoxspecies will, over time, mix with the enzyme and the mediator, althoughthis may not occur to any measurable degree over the course of theanalysis. The soluble redox species is not separated from the liquidsample by a mechanical barrier, nor is it separate from the liquidsample by virtue of its existence in a separate phase that is distinctfrom the liquid sample.

In another preferred embodiment, a soluble redox species is chosenhaving a standard reduction potential of +0.24 volts or greater, versusthe standard hydrogen electrode (SHE). In yet another preferredembodiment, a soluble redox species is chosen having a standardreduction potential of +0.35 volts or greater, versus SHE. In yetanother preferred embodiment, a redox species having a reductionpotential of about +0.48 volts versus SHE (in 0.01 M HCl) is chosen.

Thus, a wide variety of combinations of oxidoreductases, mediators, andsoluble redox species can be used to prepare an electrochemicalanalytical sensor. The use of soluble redox species having higher orlower oxidation numbers relative to their counterpart species in theredox pair is dictated by the type of reaction to be performed at theworking electrode. In one example, the analyte undergoes oxidation byinteraction with an oxidase or a dehydrogenase. In this case, the moreconcentrated redox species on the counter electrode has the higheroxidation number. A specific example of this situation is the analysisof glucose using glucose oxidase or glucose dehydrogenase. In anotherexample, the analyte undergoes reduction by interaction with areductase. In this case, the more concentrated redox species on thecounter electrode has the lower oxidation number. In either of theseexamples, the mediator may be the same substance as the moreconcentrated redox species on the counter electrode or the redox speciesof another redox pair.

If a third electrode is present in the sensor, it will also include areagent layer containing a soluble redox species as described for thecounter electrode reagent layer. Preferably the third electrode reagentlayer is identical to the counter electrode reagent layer. If thereagent layers on the third and counter electrodes are identical, thenit may be desirable simply to coat both electrodes with a single portionof a reagent layer composition.

Electrochemical sensors can be used to measure the amount of an analytethat is a substrate for the oxidoreductase on the working electrode orthat influences the reaction of the oxidoreductase with its substrate.For sensors that are intended to be operated by the patient, it isdesirable for the sample to be a small amount of a biological fluid fromthe patient. It is preferable for the sample to be analyzed directly,without the need for dilution, addition of reagents or other substances,or filtration or other methods of sample purification. Examples ofeasily obtained biological fluids include blood, urine, and saliva.Referring to FIG. 4, the sample is applied to the electrodes bydepositing a drop of the sample onto the opening provided on the inputend 60 of the strip, which is opposite the end of the strip thatconnects to the measuring unit. The sample migrates to the area betweenthe lid on top and the working electrode and the counter electrode onthe bottom. It is helpful for the lid to have an opening 54 to allow theair inside the strip to be vented upon application of the liquid sample.The area between the lid and the base may contain a substance thatretains liquid and immobilizes the sample and its contents in the areaaround the electrodes. Examples of such substances includewater-swellable polymers, such as carboxymethyl cellulose andpolyethylene glycol; and porous polymer matrices, such as dextran andpolyacrylamide.

If a sample contains a substrate for the oxidoreductase, the redoxreaction between the substrate and the enzyme can begin once the reagentlayer and the sample are in contact. The electrons produced or consumedfrom this redox reaction can be determined by applying an electricalpotential (i.e. voltage) between the working electrode and the counterelectrode, and measuring the current. The current can be correlated withthe concentration of the substrate in the sample, provided theelectrochemical strip system has been calibrated with samples containingknown amounts of substrate. The measuring unit preferably contains thenecessary circuitry and microprocessors to provide useful informationsuch as the concentration of the substrate in the sample, theconcentration of the substrate in the body of the patient, or therelevant concentration of another substance that is related to themeasured substrate.

The electrons produced or consumed by the reaction of the oxidoreductasewith its substrate are translated into a measurable current when aclosed circuit is provided by the counter electrode. The reaction thatoccurs at the counter electrode is opposite that of the reactionoccurring at the working electrode. Thus, the counter electrode suppliesor accepts electrons to the sample through the reaction of one or moreingredients of the reagent layer, depending on the type of redoxreaction occurring at the working electrode. For example, if oxidationoccurs at the working electrode, reduction occurs at the counterelectrode.

It may be desirable to delay the measuring of current in the systemuntil a given time after a voltage has been applied. Due to thecomplicated kinetic nature of the electrochemistry within the sample,the redox reactions may not reach a “steady state” in which the reactionrates have stabilized for a period of between a fraction of a second toseveral minutes. Measurements before or after this steady state has beenachieved can provide erroneous measurements of current, and thus of theconcentration of the analyte. Preferably, current measurements beginabout 20 seconds after the application of the sample.

Preferably, the voltage initially is applied to the system at the sametime as, or immediately after, the deposition of the sample. The initialapplication of voltage is maintained for 10 seconds and is then stopped,such that there is no applied voltage for a delay time of 10 seconds.After this delay time the voltage is applied again, and the current ismonitored, for a read time of 10 seconds.

For sensor strips containing a third electrode, the applied voltage canbe monitored by way of the third electrode. Any drift in the intendedvalue of the electrical potential provides feedback to the circuitrythrough the third electrode, so that the voltage can be adjustedappropriately.

The use of a third electrode may be desirable for some applications.Increased precision in the applied voltage can provide for betteraccuracy in the measurement of the analyte. When using a thirdelectrode, it may also be possible to reduce the size of the counterelectrode or to apply a smaller amount of the redox species to thecounter electrode. If the third electrode is positioned upstream of thecounter electrode, as illustrated in FIG. 14, then it may be possible todetect when insufficient sample has been applied to the strip, asituation referred to as “underfill.” Underfill detection may occur whenthere is sufficient sample to complete the circuit between the workingelectrode and the third electrode, but not to cover the counterelectrode. The lack of electrical current in the cell can be convertedelectronically into a signal to the user, instructing the user to addadditional sample to the strip.

Electrochemical sensor strips containing separately optimized reagentlayers for the working and counter electrodes can provide for improvedperformance relative to conventional sensor strips. The species of theredox pair on the counter electrode, which undergoes anoxidation-reduction reaction of opposite type with respect to thesubstrate, should be present in a larger molar ratio than the ratio of1:1 that is typically used. This larger molar ratio of the redox speciesdoes not produce significant interference with the oxidation-reductionreactions occurring near the working electrode during the time necessaryfor analysis. Also, the high concentration of the redox species providesfor a relatively stable electrochemical environment for the counterelectrode. Although the redox species is being consumed (i.e. convertedinto its counterpart species), it maintains a high enough concentrationsuch that a relatively constant linear relationship exists between themeasured current and the analyte concentration for the time scale of theanalysis.

The large molar ratio of the soluble redox species on the counterelectrode can also increase the shelf life of the sensor strip. A smalldegree of spontaneous conversion of the soluble redox species into itscounterpart species can occur during the time between the manufacture ofthe strip and its use with a sample. Since the relative concentrationwill remain high, the sensor can still produce accurate results.

EXAMPLES Example 1—Preparation of Electrode Pairs on a Base

Electrodes were formed on a base of insulating material using techniquesdescribed in U.S. Pat. Nos. 5,798,031 and 5,120,420. Referring to FIG.1, silver paste was deposited by screen printing onto a polycarbonatestrip 10. This paste was printed in a pattern 12 and 14 to form theelectrical contacts and the lower layer of the electrodes. Referring toFIG. 2, an ink containing conductive carbon and a binder was thenapplied by screen printing in a pattern 24 and 34 to form the top layerof each electrode. Referring to FIG. 3, a dielectric layer containing anacrylate-modified polyurethane was deposited onto the base and the lowerlayers of the electrodes in a pattern 40 and was then cured by exposureto UV radiation.

Example 2—Sensor Strip Having a Single, Soluble Redox Species on theCounter Electrode

A sensor strip was constructed using a pair of electrodes on a base,prepared as described in Example 1. Referring to FIG. 2, one of theelectrodes was made the working electrode by depositing onto theelectrode an aqueous mixture 26 of the enzyme glucose dehydrogenase(GDH) in combination with 20 units per microliter of coenzyme PQQ, 24 mMof 3-phenylimino-3H-phenothiazine (PIPT), 8 mM ferrocyanide, and 1% CMCpolymer. These ingredients were contained in a 100 mM phosphate bufferhaving a pH of 7.4. The other electrode was made the counter electrodeby depositing onto the electrode an aqueous mixture 36 of 200 mMferricyanide and 100 mM NaCl in a 100 mM phosphate buffer having a pH of7.4.

Referring to FIG. 4, after these aqueous mixtures were allowed to dry,the base, dielectric layer and coated electrodes were then bonded to alid 50 to form the sensor strip. The construction of the lid wasperformed as described in U.S. Pat. No. 5,798,031. A coating solution ofan aqueous polyurethane dispersion was spread on one side of apolycarbonate strip and allowed to dry. The strip was formed into a lidby embossing to form concave area 52 and by punching hole 54. The lidwas bonded to the base by aligning and contacting the lid and the base,followed by applying heat to the contact area along the periphery of thestructure.

Referring to FIG. 5, prior to being covered by the lid, the electrodestructure was imaged by optical microscopy. The working electrode is thecircle on the left, and the counter electrode is the circle on theright.

Example 3—Sensor Strip Having Two Species of a Redox Pair on the CounterElectrode

A sensor strip was constructed as in Example 2, except that the mixturedeposited to form the counter electrode contained both ferricyanide (100mM) and ferrocyanide (100 mM).

Example 4—Cyclic Voltammetric Studies of Sensor Strips

Each of the sensor strips of Examples 2-3 was analyzed by cyclicvoltammetry. The strips were re-hydrated with a 100 mM phosphate bufferhaving a pH of 7.4. Each strip was attached to an analyzer containing avoltage source and a current measuring device. The voltage was set at−0.6V, raised to +0.6V, and lowered again to −0.6V at a rate of 0.025V/sec, and the current was measured throughout the voltage cycle. A plotof the cyclic voltammograms, measuring the current as a function ofapplied voltage, for each sensor strip is shown in FIG. 6. Each of thecyclic voltammograms exhibits a peak in the positive current (oxidationpeak) due to the oxidation of the PIPT mediator. The oxidation peakoccurs near −0.2V for the strip of Example 2 and near −0.15V for thestrip of Example 3. These oxidation peaks indicate the potential atwhich a particular strip should be operated to obtain the maximumcurrent response.

Example 5—Quantification of Glucose Using Sensor Strips

The current generated as a function of glucose concentration in a samplewas studied for the sensor strips of Examples 2 and 3. Samplescontaining predetermined amounts of glucose in a 100 mM phosphate bufferhaving a pH of 7.4 were separately applied to individual sensor stripsmade according to Example 2 or according to Example 3. A potential of+0.4V was applied to the electrode pair for 10 seconds, followed by 10seconds of no applied potential. The potential of +0.4V was then appliedagain for 10 seconds, and the current was measured over during this 10second interval. FIG. 7 is a plot of the total current over themeasurement time period as a function of glucose concentration forExample 2 and Example 3.

The sensors of Example 2 showed a linear increase in average current forglucose concentrations between zero and 400 milligrams per deciliter(mg/dl). The sensors of Example 3 showed a linear increase in averagecurrent for glucose concentrations between zero and 200 mg/dL. The rangeof linear response is thus dependent on the amount of the oxidizedspecies of the redox pair, in this case ferricyanide. The non linearityof the current responses is believed to be due to a deficiency in theoxidized species of the redox pair at the counter electrode, so that theoxidation reaction at the working electrode cannot be maintained forhigher glucose concentrations. Sensors of Example 3 exhibit a linearresponse over a much smaller concentration range than the sensors ofExample 2 since the Example 3 sensors contain half as much of theoxidized species of the redox pair.

Example 6—Sensor Strip without Ferrocyanide on the Working Electrode

A sensor strip was constructed as in Example 2, except that the mixturedeposited to form the working electrode contained no ferrocyanide.

Example 7—Sensor Strip Having a Reduced Amount of the Soluble RedoxSpecies on the Working Electrode

A sensor strip was constructed as in Example 2, except that the mixturedeposited to form the working electrode contained 4 mM ferrocyanide.

Example 8—Mediator Contribution

Each of the sensor strips of Examples 2, 6 and 7 was analyzed by cyclicvoltammetry. The analysis mixtures were 40% hematocrit whole bloodcontaining no glucose. The strips were re-hydrated with 40% whole bloodsamples. Each strip was attached to an analyzer containing a voltagesource and a current measuring device. The voltage was set at −0.8V,raised to +0.6V, and lowered again to −0.8V at a rate of 0.025 V/sec,and the current was measured throughout the voltage cycle. A plot of thecyclic voltammograms, measuring the current as a function of appliedvoltage, for each sensor strip is shown in FIG. 8.

The voltammogram for the sensor of Example 6 (curve “a”) exhibited aclean oxidation peak for PIPT between −0.4V and +0.4V. A local peak inthe current around −0.3V was also exhibited in the cyclic voltammogramsof the sensors of Examples 2 (curve “c”) and 7 (curve “b”); however, themain peaks for these sensors occurred between 0V and +0.1V. All of thesepotentials are relative to the half-cell potential for the reduction offerricyanide at the counter electrode.

The oxidation peaks at positive potential is due to the oxidation offerrocyanide at the working electrode. Since the sensor of Example 2 hadtwice the molar amount of ferrocyanide on the working electrode relativeto the sensor of Example 7, the peak current due to the ferrocyanide inthe cyclic voltammogram of Example 2 is roughly twice that seen forExample 7. The cyclic voltammogram of the sensor of Example 6 showed noevidence of ferrocyanide oxidation. Since the sensor of Example 6contained no ferrocyanide on the working electrode, these resultsindicate that there is no detectable migration of ferrocyanide from thecounter electrode to the working electrode during the time scale of thisanalysis. Ferrocyanide is produced at the counter electrode by thereduction of the ferricyanide which was originally deposited on thecounter electrode. In addition, these results demonstrate that anelectrochemical biosensor of the present invention can operate with amediator on the working electrode and a species of a redox pair on thecounter electrode, where the mediator is not another species of theredox pair on the counter electrode,

Example 9—Examination of Interference of the Counter Electrode RedoxSpecies

Cyclic voltammograms of the sensor of Example 6 were performed using 40%hematocrit whole blood samples having various glucose concentrations.The voltage was set at −0.8V, raised to +0.6V, and lowered again to−0.8V at a rate of 0.025 V/sec, and the current was measured throughoutthe voltage cycle. A plot of the cyclic voltammograms, measuring thecurrent as a function of applied voltage, is illustrated in FIG. 9. Themeasured peak potential for the oxidation peak at about −0.2V becamemore positive with increased concentrations of glucose. As observed inExample 8 for the cyclic voltammetry performed in the absence ofglucose, there is no detectable migration to the working electrode ofany ferrocyanide produced at the counter electrode.

Example 10—Quantification of Glucose in Whole Blood

The current generated as a function of glucose concentration in a samplewas studied for the sensor strip of Example 6. Whole blood sampleshaving 40% hematocrit were analyzed for glucose concentration using aYSI glucose analyzer as the reference method. Once the actual glucoseconcentrations of the various samples were determined, the samples wereapplied separately to individual sensor strips made according to Example6. A potential of +0.4V was applied to the electrode pair for 10seconds, followed by 10 seconds of no applied potential. The potentialof +0.4V was then applied again for 10 seconds, and the current wasmeasured over during this 10 second interval. FIG. 10 is a plot of thetotal current over the measurement time period as a function of glucoseconcentration for Example 6. The sensors of Examples 6 showed a linearresponse in current as a function of blood glucose concentration fromzero to about 600 mg/dL.

Example 11—Sensor Strip with PIPT on Working and Counter Electrode

A sensor strip was constructed as in Example 1, except that the mixturedeposited on the counter electrode contained 25 mM PIPT. Sensor stripsmade in this way were analyzed by cyclic voltammetry in 100 mM phosphatebuffer mixtures at pH 7.4, where the analysis mixtures had variousglucose concentrations. The potential was cycled between −0.4V and +0.6Vat a rate of 0.025 V/sec. The cyclic voltammograms for these strips areillustrated in FIG. 11.

Since the oxidation of PIPT is measured relative to its own redoxpotential at the counter electrode, the oxidation/reduction peaks arecentered close to 0 volt. On the other hand, the oxidation peak of theferrocyanide on the working electrode shifted to a higher oxidationpotential relative to the PIPT peak. These studies showed thefeasibility of using PIPT as the redox species on the counter electrode.The working electrode could contain PIPT, as analyzed in this example,or it could contain ferricyanide as the mediator.

Example 12—Preparation of Sensor Strips Containing Three Electrodes

Electrodes are formed on a base of insulating material as described inExample 1, except that a third electrode is also applied to the base.Referring to FIGS. 12-14, silver paste is deposited by screen printingonto a polycarbonate strip 10. This paste is printed in a pattern 12, 13and 14 to form the electrical contacts and the lower layer of theelectrodes. An ink containing conductive carbon and a binder is thenapplied by screen printing in a pattern 24, 34 and 74 to form the toplayer of each electrode. A dielectric layer containing anacrylate-modified polyurethane is deposited onto the base and the lowerlayers of the electrodes in a pattern to expose the electrodes and isthen cured by exposure to UV radiation.

One of the electrodes is made the working electrode by depositing ontothe electrode an aqueous mixture 26 of the enzyme glucose dehydrogenase(GDH) in combination with 20 units per microliter of coenzyme PQQ, 24 mMof 3-phenylimino-3H-phenothiazine (PIPT), and 1% CMC polymer. Theseingredients are contained in a 100 mM phosphate buffer having a pH of7.4.

One of the other electrodes is made the counter electrode by depositingonto the electrode an aqueous mixture 36 of 200 mM ferricyanide and 100mM NaCl in a 100 mM phosphate buffer having a pH of 7.4. The thirdelectrode is made by covering the remaining electrode with a reagentlayer 76. The deposition of the reagent layer 76 may be done as aseparate step with a third aqueous mixture, or the reagent layer 76 maybe applied as a portion of the same aqueous mixture deposited to makethe counter electrode.

After these aqueous mixtures are dried, the base, dielectric layer andelectrodes are then bonded to a lid to form the sensor strip. A coatingsolution of an aqueous polyurethane dispersion is spread on one side ofa polycarbonate strip and allowed to dry. The strip is formed into a lidby embossing to form a concave area and by punching a hole. The lid isbonded to the base by aligning and contacting the lid and the base,followed by applying heat to the contact area along the periphery of thestructure.

Although the invention has been described and illustrated with referenceto specific illustrative embodiments thereof, it is not intended thatthe invention be limited to those illustrative embodiments. Thoseskilled in the art will recognize that variations and modifications canbe made without departing from the true scope and spirit of theinvention as defined by the claims that follow. It is therefore intendedto include within the invention all such variations and modifications asfall within the scope of the appended claims and equivalents thereof.

1-22. (canceled)
 23. A method of making an electrochemical sensor strip,the method comprising: depositing a first electrode on a base;depositing a second electrode on the base; applying a first reagentlayer onto the first electrode, the first reagent layer comprising anelectroactive organic molecule and an oxidoreductase configured tofacilitate a redox reaction of an analyte; and applying a second reagentlayer onto the second electrode, the second reagent layer consistingessentially of a redox pair comprising a first soluble redox species anda second soluble redox species, the redox pair being selected from agroup consisting of an organotransition metal complex, a transitionmetal coordination complex, and mixtures thereof.
 24. The method ofclaim 23, wherein the first soluble redox species comprises at least oneof ruthenium(II) hexaamine or ruthenium(III) hexaamine.
 25. The methodof claim 23, wherein the electroactive organic molecule includescoenzyme pyrroloquinoline quinone, substituted benzoquinones,substituted naphthoquinones, N-oxides, nitroso compounds,hydroxylamines, oxines, flavins, phenazines, phenothiazines,indophenols, indamines, phenazinium salts, phenoxazinium salts,3-phenylimino-3H-phenothiazines, or 3-phenylimino-3H-phenoxazines, orany mixture thereof.
 26. The method of claim 23, wherein theelectroactive organic molecule includes at least one of a3-phenylimino-3H-phenothiazine or a 3-phenylimino-3H-phenoxazine. 27.The method of claim 23, wherein the analyte comprises glucose.
 28. Themethod of claim 27, wherein the oxidoreductase comprises glucoseoxidase, glucose dehydrogenase, or a combination thereof.
 29. The methodof claim 23, further comprising covering a portion of the base with adielectric layer such that the first and second reagent layers areexposed.
 30. The method of claim 23, further comprising mating a lid tothe base such that the lid is over the first and second electrodes andthe first and second reagent layers.
 31. The method of claim 23, whereinthe depositing of the second electrode comprises depositing a pattern ofa non-ionizing conductive material.
 32. The method of claim 23, furthercomprising: depositing a third electrode on the base; and applying athird reagent layer onto the third electrode, the third reagent layercomprising a third soluble redox species.
 33. The method of claim 32,wherein the third soluble redox species is substantially identical tothe first soluble redox species of the second reagent layer.
 34. Themethod of claim 32, wherein the composition of the third reagent layeris substantially identical to the composition of the second reagentlayer.
 35. The method of claim 23, wherein the first soluble redoxspecies has a standard reduction potential of at least about +0.24volts.
 36. A method of making an electrochemical sensor strip, themethod comprising: forming a first connecting conductor on a base, thefirst connecting conductor extending to a proximal edge of the base, andthe base being formed of an insulating material; forming a secondconnecting conductor on the base, the second connecting conductorextending to the proximal edge of the base; forming a first mainconductor on the base contiguous to a distal end of the first connectingconductor; forming a second main conductor on the base contiguous to adistal end of the second connecting conductor; applying a first reagentlayer onto the first main conductor, the first reagent layer comprisingan electroactive organic molecule and an oxidoreductase configured tofacilitate a redox reaction of an analyte; and applying a second reagentlayer onto the second main conductor, the second reagent layerconsisting essentially of a redox pair comprising a first soluble redoxspecies and a second soluble redox species, the redox pair beingselected from a group consisting of an organotransition metal complex, atransition metal coordination complex, and mixtures thereof.
 37. Themethod of claim 36, wherein the first connecting conductor and the firstmain conductor are formed of a first continuous strip of metal foil, andthe second connecting conductor and the second main conductor are formedof a second continuous strip of metal foil.
 38. The method of claim 37,further comprising: forming a first surface conductor on the first mainconductor and below the first reagent layer; and forming a secondsurface conductor on the second main conductor and below the secondreagent layer.
 39. The method of claim 38, wherein the first surfaceconductor and the second surface conductor are formed of conductivecarbon.
 40. The method of claim 36, further comprising: forming a thirdconnecting conductor on the base, the third connecting conductorextending to the proximal edge of the base; forming a third mainconductor on the base contiguous to a distal end of the third connectingconductor; and applying a third reagent layer onto the third mainconductor, the third reagent layer comprising a third soluble redoxspecies.
 41. The method of claim 40, further comprising forming a thirdsurface conductor on the third main conductor and below the thirdreagent layer.
 42. The method of claim 41, wherein the third connectingconductor and the third main conductor are formed of a continuous stripof metal foil, and the third surface conductor is formed of conductivecarbon.